Stabilized enzymes and detergent compositions

ABSTRACT

The invention relates to stabilized subtilisin proteases wherein one or more prolines is substituted for a native amino acid at stabilizing positions defined both by the range of the dihedral angles in the primary structure present at the substitution site and by selected characteristics of the protease secondary structure in the vicinity of the substitution site, to nucleotide sequences that encode the stabilized proteases, and to host organisms that contain the nucleotide sequences encoding the stabilized proteases.

TECHNICAL FIELD

This invention relates to novel stabilized proteases, nucleotidesequences encoding the stabilized proteases, and host organismscontaining the nucleotide sequences encoding the novel stabilizedproteases.

BACKGROUND ART

Proteases/Subtilisins

Proteases, or (interchangeably) peptidases, are enzymes that cleave theamide linkages in protein substrates. Bacteria of the Bacillus speciessecrete two extracellular species of protease, a neutral ormetalloprotease, and an alkaline protease which is functionally a serineendopeptidase, referred to as subtilisin.

A serine protease is an enzyme which catalyses the hydrolysis of peptidebonds, and in which there is an essential serine residue at the activesite White, Handler and Smith (1973); Principles of Biochemistry; FifthEdition, McGraw-Hill Book Company, N.Y., 271-272!.

The bacterial serine proteases have molecular weights in the range of20,000 to 45,000. They hydrolyse simple terminal esters and are similarin activity to eukaryotic chymotrypsin, also a serine protease. A morenarrow term, alkaline protease, covering a sub-group, reflects the highpH optimum of some of the serine proteases, from pH 9.0 to 11.0 forreview, see Priest; Bacteriological Rev., 41 711-753 (1977)!.

In relation to the present invention a subtilisin is a serine proteaseproduced by Gram-positive bacteria or fungi. According to anotherdefinition, a subtilisin is a serine protease, wherein the relativeorder of the amino acid residues in the catalytic triad is Asp--His--Ser(positions 32, 64, and 221). A wide variety of subtilisins has beenidentified, and the amino acid sequences of a number of subtilisins havebeen determined. These include among others six subtilisins fromBacillus strains, namely, subtilisin 168, subtilisin BPN', subtilisinCarlsberg, subtilisin DY, subtilisin amylosacchariticus, andmesentericopeptidase Kurihara et al. (1972); J. Biol. Chem.; 2475629-5631; Wells et al. (1983); Nucleic Acids Res.; 11 7911-7925; Stahland Ferrari (1984); J. Bacteriol.; 159 811-819; Jacobs et al. (1985);Nucl. Acids Res.; 13 8913-8926; Nedkov et al. (1985); Biol. Chem.Hoppe-Seyler; 366 421-430; Svendsen et al. (1985); FEBS LETTERS; 196228-232! one subtilisin from an actino-mycetales, Thermitase fromThermoactinomyces vulgaris Meloun et al. (1985); FEBS LETTERS; 1983195-200!, and one fungal subtilisin, proteinase K from Tritirachiumalbum Jany and Mayer (1985); Biol. Chem. Hoppe-Seyler; 366 584-492!.

Proteases such as subtilisins have found much utility in industry,particularly in detergent formulations, as they are useful for removingproteinaceous stains.

The Structure of Proteins

Proteases are globular proteins and quite compact due to theconsiderable amount of folding of the long polypeptide chain. Thepolypeptide chain essentially consists of the "backbone" and its"side-groups". As the peptide bond is planar, only rotations around theC_(a) --N axis and the C_(a) --C' axis are permitted. Rotation aroundthe C_(a) --N bond of the peptide backbone is denoted by the torsionangle φ (phi), rotation around the C_(a) --C' bond by ψ (psi) vide e.g.Creighton, T. E. (1984); Proteins; W. H. Freeman and Company, New York!.The choice of the values of these angles of rotation is made byassigning the maximum value of +180° (which is identical to -180°) tothe maximally extended chain. In the fully extended polypeptide chain,the N, C_(a) and C' atoms are all "trans" to each other. In the "cis"configuration, the angles φ and ψ are assigned the value of 0°. Rotationfrom this position around the bonds so that the atoms viewed behind therotated bond move "counter-clockwise" is assigned negative values bydefinition, those "clockwise" are assigned positive values. Thus, thevalues of the torsion angles lie within the range -180° to +180°.

Since the C_(a) -atoms are the swivel point for the chain, theside-groups (R-groups) associated with the C_(a) -atoms become extremelyimportant with respect to the conformation of the molecule.

The term "conformation" defines the participation of the secondary andtertiary structures of the polypeptide chains in moulding the overallstructure of a protein. The correct conformation of a protein is ofprime importance to the specific structure of a protein and contributesgreatly to the unique catalytic properties (i.e. activity andspecificity) of enzymes and their stability.

The amino acids of polypeptides can be divided into four general groups:nonpolar, uncharged polar, and negatively or positively charged polaramino acids. A protein molecule, when submerged in its aqueousenvironment in which it normally occurs, tends to expose a maximumnumber of its polar side-groups to the surrounding environment, while amajority of its nonpolar side groups is oriented internally. Orientationof the side-groups in this manner leads to a stabilization of proteinconformation.

Proteins, thus, exist in a dynamic equilibrium between a folded andordered state, and an unfolded and disordered state. This equilibrium inpart reflects the short range interactions among the different segmentsof the polypeptide chain, which tends to stabilize the overall structureof proteins. Thermodynamic forces simultaneously tend to promoterandomization of the unfolding molecule.

A way to engineer stabilized proteins is to reduce the extent ofunfolding by decreasing the flexibility of the polypeptide backbone, andsimultaneously decreasing the entropy of the unfolded chain. So far onlyfew attempts have been made to implement this rationale in thedevelopment of novel stabilized proteases.

A general principle of increasing protein thermostability has beenprovided Suzuki, Y. (1989); Proc. Japan Acad.; 65 Ser. B!. In thisarticle Suzuki states that the thermostability of a globular protein canbe enhanced cumulatively to a great extent by increasing the frequencyof proline occurrence at the second site of β-turns without significantalterations in the secondary and tertiary structures as well as in thecatalytic function of enzymes. The principle is based on various factsand findings, among these the fact that proline residues show a strongtendency to occur preferentially at the second site of β-turns Levitt, M(1978); Biochemistry; 17 4277-4285; and Chou, P. Y. & Fasman, G. D.(1977); J. Mol. Biol.; 115 135-175!. The principle is restricted toinsertion of proline into the second site of β-turns in proteins, noother sites are mentioned.

International Patent Publication WO 89/01520 (Cetus Corporation, USA)provides a method for increasing the stability of a protein bydecreasing the configurational entropy of unfolding the protein. Themethod is applied on a Streptomyces rubiqinosus xylose isomerase, and itinvolves substitution of an amino acid with proline, or replacement ofglycine with alanine, at predicted substitution sites.

In International Patent Publication WO 89/09819 (Genex Corporation, USA)a method for combining mutations for stabilization of subtilisins isprovided. This publication lists a number of amino acid mutations thathave been found to be thermally stabilizing mutations. The listcomprises substitution of serine with proline at position 188 ofsubtilisins (BPN' numbering).

International Patent Publication WO 87/05050 (Genex Corporation, USA)describes a method for mutagenesis and screening. By this method one ormore mutations are introduced by treatment with mutagenizing agents, andthe method includes subsequent screening for products with alteredproperties. As a result of this random mutagenesis a subtilisin with aproline residue at position 188 (BPN' numbering) is provided.

It is an object of this invention to provide novel proteases havingimproved stability.

SUMMARY OF THE INVENTION

The present invention provides novel stabilized proteases, in which anaturally occurring amino acid residue (other than proline) has beensubstituted with a proline residue at one or more positions, at whichposition(s) the dihedral angles φ (phi) and ψ (psi) constitute valueswithin the intervals -90°<φ<-40° and -180°<ψ<180°!, preferably withinthe intervals -90°<φ<-40° and 120°<ψ<180°! or -90°<φ<-40° and-50°<ψ<10°!, and which position(s) is/are not located in regions, inwhich the protease is characterized by possessing α-helical or, β-sheetstructure.

In another aspect, the invention relates to nucleotide sequencesencoding the proteases. In further aspects, the invention relates to anexpression vector comprising a nucleotide sequence encoding a protease,and to a host organism containing this expression vector.

Subtilisins

In the context of this invention a subtilisin is defined as a serineprotease produced by gram-positive bacteria or fungi. According toanother definition, a subtilisin is a serine protease, wherein therelative order of the amino acid residues in the catalytic triad isAsp--His--Ser (positions 32, 64, and 221, BPN' numbering).

Amino Acids

As abbreviations for amino acids the following symbols are used:

    ______________________________________    A        =       Ala     =     Alanine    C        =       Cys     =     Cysteine    D        =       Asp     =     Aspartic acid    E        =       Glu     =     Glutamic acid    F        =       Phe     =     Phenylalanine    G        =       Gly     =     Glycine    H        =       His     =     Histidine    I        =       IIe     =     Isoleucine    K        =       Lys     =     Lysine    L        =       Leu     =     Leucine    M        =       Met     =     Methionine    N        =       Asn     =     Asparagine    P        =       Pro     =     Proline    Q        =       Gln     =     Glutamine    R        =       Arg     =     Arginine    S        =       Ser     =     Serine    T        =       Thr     =     Threonine    V        =       Val     =     Valine    W        =       Trp     =     Tryptophan    Y        =       Tyr     =     Tyrosine    B        =       Asx     =     Asp (D) or Asn (N)    Z        =       Glx     =     Glu (E) or Gln (Q)    X        =       an arbitrary amino acid    *        =       deletion or absent amino acid    ______________________________________

Protease Variants

A stabilized protease of this invention is a protease variant or mutatedprotease. By a protease variant or mutated protease is meant a proteaseobtainable by alteration of a DNA nucleotide sequence of the parent geneor its derivatives. The protease variant or mutated protease may beexpressed and produced when the DNA nucleotide sequence encoding theprotease is inserted into a suitable vector in a suitable host organism.The host organism is not necessarily identical to the organism fromwhich the parent gene originated.

Amino Acid Numbering

In the context of this invention a specific numbering of amino acidresidue positions in subtilisins is employed. By alignment of the aminoacid sequences of various subtilisins along with subtilisin BPN', it ispossible to allot a number to the amino acid residue position in anysubtilisin to the number of the analogous amino acid position insubtilisin BPN' ("BPN' numbering", vide e.g. International PatentPublications Nos. WO 89/06279 and WO 91/00345).

In describing the various protease variants produced or contemplatedaccording to the invention, the following nomenclatures were adapted forease of reference:

Original amino acid; Position; Substituted amino acid!

Accordingly, the substitution of alanine with proline in position 195 isdesignated as:

A195P

Deletion of an aspartic acid at position 36 is indicated as: D36*, andan insertion in such a position is indicated as: 36D for insertion of anaspartic acid in position 36.

Multiple mutations are separated by plusses, i.e.:

A194P+G195E

representing mutations in positions 194 and 195 substituting alaninewith proline and glycine with glutamic acid, respectively.

If a substitution is made by mutation in e.g. subtilisin 309, theproduct is designated e.g. "subtilisin 309/G195E".

All positions mentioned in this context refer to the BPN' numbersdescribed above.

Proteolytic Activity

In the context of this invention proteolytic activity is expressed inKilo NOVO Protease Units (KNPU). The activity is determined relativelyto an enzyme standard (SAVINASE™), and the determination is based on thedigestion of a dimethyl casein (DMC) solution by the proteolytic enzymeat standard conditions, i.e. 50° C., pH 8.3, 9 min. reaction time, 3min. measuring time. A folder AF 220/1 is available upon request to NovoNordisk A/S, Denmark, which folder is hereby included by reference.

Wash Performance

The ability of an enzyme to catalyse the degradation of variousnaturally occurring substrates present on the objects to be cleanedduring e.g. wash is often referred to as its washing ability,washability, detergency, or wash performance. Throughout thisapplication the term wash performance will be used to encompass thisproperty.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to theaccompanying drawings, in which:

FIG. 1 (sheets 1/20-15/20) shows the construction of a synthetic gene;and

FIG. 2 (sheets 16/20-20/20) shows the residual activity of subtilisin309 variants compared to wild type enzyme after storage in a liquiddetergent.

DETAILED DISCLOSURE OF THE INVENTION

The present invention provides novel stabilized proteases, in which anaturally occurring amino acid residue (other than proline) has beensubstituted with a proline residue at one or more positions, at whichposition(s) the dihedral angles φ (phi) and ψ (psi) constitute values inthe intervals -90°<φ<-40° and -180°<ψ<180°!, preferably the intervals-90°<φ<-40° and 120°<ψ<180°! or -90°φ<-40° and -50°<ψ<10°!, and whichposition(s) is/are not located in regions, in which the protease ischaracterized by possessing α-helical or β-sheet structure.

In the context of this invention, a stabilized protease is a proteasevariant or mutated protease, being functionally equivalent or havingstructural features similar to a naturally occurring protease, and inwhich protease a naturally occurring amino acid residue (other thanproline) has been substituted with a proline residue at one or morepositions, at which position(s) the dihedral angles φ (phi) and ψ (psi)constitute values within the intervals -90°<φ<-40° and -180°<ψ<180°!,preferably the intervals -90°<φ<-40° and 120°<ψ<180°! or -90°<φ<-40° and-50°<ψ<10°!, and which position(s) is/are not located in regions, inwhich the protease is characterized by possessing α-helical or β-sheetstructure.

Moreover, in the context of this invention, a stabilized protease is aprotease having improved stability, e.g. in respect to thermalstability, storage stability, etc., when compared to the parent enzyme.

Defining Secondary Structure of Proteins

The stabilized proteases of the invention may be obtained by subjectingthe protease in question to analysis for secondary structure,identifying residues in the protease having dihedral angles φ (phi) andψ (psi) confined to the intervals -90°<φ<-40° and -180°<ψ<180°!,preferably the intervals -90°<φ<-40° and 120°<ψ<180°! or -90°<φ<-40° and-50°<ψ<10°!, excluding residues located in regions in which the proteaseis characterized by possessing α-helical or β-sheet structure, if aproline residue is not already at the identified position(s),substitution of the naturally occurring amino acid residue with aproline residue at the identified position(s), preferably by sitedirected mutagenesis applied on a gene encoding the protease inquestion, and gene expression by insertion of the gene encoding thestabilized protease in a suitable host organism, followed by cultivationof said host organism in a suitable nutrient medium, and recovery of thedesired protease.

This preparation includes subjecting the protease in question toanalysis for secondary structure. To perform such analysis the atomicstructure of the protease has to be elucidated. The atomic structure canbe determined by X-ray diffraction techniques. X-ray diffractiontechniques are described by e.g. Hendrickson, W. A. X-ray diffraction;in Protein Engineering (Ed: Oxender, D. L. and Fox, C. F.), ch. 1; AlanR. Liss, Inc. (1987)! and Creighton, T. E., supra, ch. 6.

The crystal structure of Subtilisin 309 has been deduced vide Beizel,B., Klupsch, S., Papendorf, G., Hastrup, S., Branner, S., and Wilson,K.S. (1992); J. Mol. Biol. 223 427-445!, and the coordinates have beendeposited and are available from the Brookhaven Protein Data BankBemstein et al. (1977); J. Mol. Biol. 112 535-542!.

When the atomic structure has been determined, it is possible to computedihedral angles from the atomic coordinates. Moreover, it is possible toassign secondary structure elements. The secondary structure elementsare defined on the basis of hydrogen bindings. Cooperative secondarystructure is recognized as repeats of the elementary hydrogen-bondingpatterns "turn" and "bridge". Repeating turns are "helices", repeatingbridges are "ladders", connected ladders are "sheets".

Analysis for secondary structure elements requires a computerizedcompilation of structure assignments and geometrical features extractedfrom atomic coordinates. The conventional method to elucidate thesecondary structure of a protein, based on its atomic coordinates, isdescribed by Kabsch, W. and Sander, C. Biopolymers (1983) 22 2577-2637!.In this article an algorithm for extracting structural features from theatomic coordinates by a pattern-recognition process is provided. First,H-bonds are identified based on electrostatic interactions between pairsof H-bonding groups. Next, the patterns of H-bonding are used to definesecondary structure elements such as turns M, bends (S), bridges (B),helices (G,H,I), β-ladders (E) and β-sheets (E).

A computer program DSSP (Define Secondary Structure of Proteins),enabling the computation of Kabsch & Sander files and written instandard PASCAL, is available from the Protein Data Bank, ChemistryDept., Brookhaven National Laboratory, Upton, N.Y. 11973.

After the dihedral angles φ (phi) and ψ (psi) for the amino acids havebeen calculated, based on the atomic structure in the crystallineproteases, it is possible to select position(s) which has/have dihedralphi and psi angles favourable for substitution with a proline residue.The aliphatic side chain of proline residues is bonded covalently to thenitrogen atom of the peptide group. The resulting cyclic five-memberedring consequently imposes a rigid constraint on the rotation about theN--C_(a) bond of the peptide backbone and simultaneously prevents theformation of hydrogen bonding to the backbone N-atom. For thesestructural reasons, prolines are generally not compatible with α-helicaland β-sheet secondary conformations. Due to the same rotationalconstraint about the C_(a) --N bond, and due to the requirement thatneighbouring amino acids in the chain are not perturbed, the magnitudesof the dihedral angles phi and psi (and in particular phi) are confinedto limited intervals for proline residues in polypeptides. The dihedralangles for proline residues in polypeptides are almost exclusivelywithin the intervals -90°<φ<-40° and -180°<ψ<180°!, preferably theintervals -90°<φ<-40° and 120°<ψ<180°! or -90°<φ<-40° and -50°<ψ<10°!.In this context, both cis- and trans-proline residues are considered.

A proline residue may already occur at one or more positions pointed outby the procedure described above, and then a substitution is, of course,irrelevant. Otherwise, the method includes substitution of the naturallyoccurring amino acid residue with a proline residue.

However, a substitution into proline at every of the predicted positionsmay not always bring about improved thermostability of the protease. Atsome of the positions revealed by this method, a substitution of anaturally occurring amino acid residue into a proline residue may evencause destabilisation due to unpredictable factors, such as loss ofessential flexibility, loss of H-bond possibilities, unpredictablesterical hindrance, etc. Such "critical sites" are not to be foreseen.

It is to be expected that the stabilizing (or destabilizing) effects ofindividual substitutions are additive, vide e.g. Wells, J. A.Biochemistry (1990) 29 (37) 8510-8517! and Table 4, below.

If a subtilisin different from subtilisin 309 is subjected to thismethod (although the two subtilisins may seem very much similar), notnecessarily the same number of positions, nor particularly the identicalpositions, may result from the method. It is likely that some of thepositions may be identical, and it is likely that the numbers ofpositions are of equal magnitude, but it is not to be foreseen.

However, it seems likely that the stabilizing proline substitutionsresulting from the above described method, applied to any specificprotease, may also have a stabilizing effect on any other protease,independent of the result of the above described method applied to sucha protease.

When performing the method on a subtilisin 309 molecule (videInternational Patent Application No. PCT/DK88/00002), a set of data aslisted in Tables 1 and 2 can be obtained. Table 1 depicts a set ofpositions that--with respect to phi and psi angles--meet one criterion,and Table 2 depicts an additional set of positions that meet anothercriterion.

                  TABLE 1    ______________________________________    Proline Mutants Proposed in Subtilisin 309 Based on phi and psi Angles.    Criteria:           -90° < phi < -40° and 120° < psi           < 180°, or           -90° < phi < -40° and -50° < psi           < 10°.           Neither part of an alpha helix nor a beta sheet structure.    BPN'   phi    phi    Amino Struc-    numbers           angle  angle  acid  ture  Mutant & Comments    ______________________________________    5      -59    148    P           Pro already in Subt.309    11     -80    -1     V     T    19     -85    1      R     T    24     -57    132    S     T    33     -79    10     T     S    37     -89    145    S    38     -58    146    T    40     -60    -27    P     T     Pro already in Subt.309    52     -59    131    P     T     Pro already in Subt.309    55     -71    -22    P           Pro already in Subt.309    57     -90    167    S     S    59     -60    151    Q    74     -57    141    A    75     -67    148    L    83     -81    173    G    84     -72    -35    V     S    86     -59    -12    P     T     Pro already in Subt.309    88     -68    152    A    97     -73    174    G    98     -57    -32    A     T    99     -67    -12    S     T    119    -70    149    M    129    -68    -24    P     S     Pro already in Subt.309    130    -85    126    S     S    131    -63    156    P           Pro already in Subt.309    145    -88    10     R     T    147    -86    134    V    153    -74    -20    S    156    -82    -11    S     S    158    -70    159    A    163    -66    166    S    169    -52    -33    A     T    170    -66    -21    R     T    171    -67    141    Y     S    172    -52    -43    A     T    173    -79    1      N     T    182    -63    -16    Q     T    186    -65    133    R     B    187    -65    151    A    188    -64    -27    S     T    189    -77    -17    F     T    191    -69    151    Q    194    -56    130    A     T    210    -53    155    P     T     Pro already in Subt.309    239    -58    -25    P     T     Pro already in Subt.309    241    -79    149    W    242    -86    178    S    254    -71    165    A     S    256    -59    136    S    259    -48    131    S    265    -69    -20    S     T    267    -79    127    L     B    268    -58    141    V    ______________________________________

By relaxing the constraint on the psi angle relative to the criteria setup in Table 1, four additional mutants are proposed, vide Table

                  TABLE 2    ______________________________________    Proline Mutants Proposed in Subtilisin 309 based on phi and psi Angles.    Relaxed criteria:                -90° < phi < -40 and -180° < psi < 180.                Neither part of an alpha helix nor a beta sheet                structure.    BPN'   phi     psi    Amino Struc-    numbers           angle   angle  add   ture   Mutant & Comments    ______________________________________     76    -89     102    N     S    125    -90     67     S    160    -83     27     G     S    255    -89     111    T     B    ______________________________________

Preferred Stabilized Proteases

Preferably, a protease of the invention is a stabilized subtilisinprotease. In a more specific aspect, a preferred protease of theinvention is a subtilisin, in which the stabilized subtilisin obtainedis any subtilisin which comprises a substitution into a proline residueat one or more of the positions listed in Tables 1 and 2 (BPN' numbers),or positions analogous hereto.

In a more specific aspect, a proteases of the invention is a subtilisinin which a naturally occurring amino acid (other than proline) has beensubstituted with a proline residue at one or more of the positions: 38,57, 98, 172, 188, 194, 242, and 259 (BPN' numbers). In further preferredembodiments, the subtilisin in addition comprises one or more of thefollowing substitutions: 27R, 36D, 76D, 97N, 98R, 104Y, 120D, 128G,195E, 206C, 218S, 235L, 235R, 237R, 251E, and 263F (BPN' numbers).

In a yet more specific aspect, the protease of the invention is astabilized subtilisin 309, a stabilized subtilisin 147, a stabilizedsubtilisin BPN', or a stabilized subtilisin Carlsberg. Subtilisin 309and Subtilisin 147 are variants of Bacillus lentus and described in U.S.Pat. No. 3,723,250 and International Patent Application PCT/DK88/00002,Subtilisin BPN' is described by Wells et al. Nucleic Acids Res. (1983)11 7911-7925!; Subtilisin Carlsberg is described by Smith et al. Smith,E. L.; DeLange, R. J.; Evans, W. H.; Landon, W.; Markland, F. S. (1968);Subtilisin Carlsberg V. The complete sequence: comparison withsubtilisin BPN'; Evolutionary relationships.; J. Biol. Chem. 243 (9)2184-2191), and Jacobs et al. Nucl. Acids Res. (1985) 13 8913-8926!.

In another specific aspect, a protease of the invention is a stabilizedsubtilisin 309 in which one or more of the following substitutions havebeen introduced: T38P, S57P, A98P, S188P, A172P, A194P, S242P, and S259P(BPN' numbers). In further preferred embodiments, this subtilisin inaddition comprises one or more of the following substitutions: K27R,*36D, N76D, G97N, A98R, V104Y, H1 20D, S128G, G195E, Q206C, N218S,K235L, K235R, K237R, K251E, and Y263F (BPN' numbers).

In yet another preferred embodiment, the protease of the invention is asubtilisin 309, in which one or more of the following substitutions havebeen introduced: T38P, S57P, A98P, A172P, A194P, S242P, and S259P (BPN'numbers). In a further preferred embodiment, this subtilisin 309 inaddition comprises one or more of the following substitutions: K27R,*36D, N76D, G97N, A98R, V104Y, H120D, S128G, G195E, Q206C, N218S, K235L,K235R, K237R, K251E, and Y263F (BPN' numbers).

Most preferred proteases of the invention are: Subtilisin309/K27R+*36D+G97N+A98R+A194P+K235R+K237R+K251E+Y263F, Subtilisin309/K27R+*36D+G97N+A194P+K235R+K237R+K251E+Y263F, Subtilisin309/K27R+*36D+G97N+A194P+K235R+K237R+Y263F, Subtilisin309/*36D+N76D+H120D+A194P+G195E+K235L, Subtilisin309/*36D+G97N+V104Y+H120D+A194P+G195E, Subtilisin309/*36D+G97N+V104Y+H120D+A194P+G195E+K235L, Subtilisin309/*36D+G97N+H120D+A194P+G195E, Subtilisin 309/*36D+G97N+H120D+A194P+G195E+K235L, Subtilisin 309/*36D+V104Y+H120D+A194P+G195E,Subtilisin 309/*36D+V104Y+H120D+A194P+G195E+K235L, Subtilisin 309/*36D+H120D+A194P+G195E, Subtilisin 309/*36D+H120D+A194P+G195E+K235LandSubtilisin 309/A194P (BPN' numbers).

The Effect of Proline Stabilization

The purified variants obtained according to this invention have beentested to wash at least equally well compared to the wild-typesubtilisin (subtilisin 309), vide Example 5 for experimental data.

The storage stability generally reflects the thermostability, i.e.improved thermostability corresponds with improved storage stability.The improvement in thermostability of purified variants has been testedby a differential scanning calorimetry (DSC) method, vide Example 3 forexperimental data. The result of these tests is shown in Table 3, below.The storage stability has been tested by a Mini Storage Test, videExample 4 for experimental data, and the results of the storagestability test are shown in FIG. 2.

                  TABLE 3    ______________________________________    Stabilization relative to Subtilisin 309                Relative stabilization                Δ DSC    Variant     (°C.)    ______________________________________    S24P        -2.0° C.    T38P        -0.1° C.    S57P        0.7° C.    A98P        0.1° C.    S99P        -0.2° C.    A172P       0.1° C.    Q182P       -2.5° C.    S188P       1.5° C.    A194P       2.6° C.    S242P       1.4° C.    S256P       -2.1° C.    S259P       0.6° C.    ______________________________________

It appears from Table 3 that 3 of the variants constructed possesssignificantly improved thermostability, 2 of the variants possess onlyslightly improved thermostability, 4 of the variants possess nosignificant change in thermostability, and 3 of the variants possessignificantly decreased thermostability, when compared to the wild-typeenzyme.

These results demonstrate that although a clear rationale exists forstabilization by introduction of proline residues into a protein by thePhi-Psi-Concept described in this specification, no conclusion as to thestabilizing effect of the individual variants is predictable.

However, when a variant with improved thermostability has been obtained,the stabilizing effect of individual mutations in general is consideredadditive. This is demonstrated in Table 4 below, where the thermaldenaturation temperatures, as measured by differential scanningcalorimetry (DSC), of Subtilisin 309 variants relative to the wild-typeenzyme are presented.

Thus, stabilized protease variants which, further to the stabilizingproline residue(s) inserted, contain one or more additional stabilizingmutations as described in this specification, are considered within thescope of this invention.

                                      TABLE 4    __________________________________________________________________________    Additive Stabilizing Effect in Subtilisin 309 Variants    Figures in parenthesis indicate the sum of Δ DSC temperatures for    variants tested individually.                                   Δ DSC    Variants                       (°C.)    __________________________________________________________________________    G195E                          0.1        H120D                      0.2            K235L                  0.0                *36D               4.0                   N76D            4.3                       A194P       2.6                           G97N    -0.3                               V104Y                                   2.3    G195E        H120D            K235L                  0.3 (0.3)    G195E        H120D            K235L                *36D               4.0 (4.3)    G195E        H120D            K235L                *36D   A194P       7.0 (6.9)    G195E        H120D            K235L                *36D                   N76D            7.0 (8.6)    G195E        H120D            K235L                *36D                   N76D                       A194P       10.4                                       (11.2)    G195E        H120D   *36D   A194P                           G97N    7.4 (6.6)    G195E        H120D   *36D   A194P                           G97N                               V104Y                                   8.2 (8.2)    G195E        H120D            K235L                *36D   A194P                           G97N    6.9 (6.6)    G195E        H120D            K235L                *36D   A194P                           G97N                               V104Y                                   8.0 (8.9)    __________________________________________________________________________

Method For Producing Mutations In Genes Encoding Proteases

Many methods for introducing mutations into genes are well known in theart. After a brief discussion of cloning subtilisin genes, methods forgenerating mutations at specific sites within the subtilisin gene willbe discussed.

Cloning A Subtilisin Gene

The gene encoding subtilisin may be cloned from any Gram-positivebacteria or fungus by various methods, well known in the art. First agenomic, and/or cDNA library of DNA must be constructed usingchromosomal DNA or messenger RNA from the organism that produces thesubtilisin to be studied. Then, if the amino-acid sequence of thesubtilisin is known, homologous, oligonucleotide probes may besynthesized, labelled, and used to identify subtilisin-encoding clonesfrom a genomic library of bacterial DNA, or from a fungal cDNA library.Alternatively, a labelled oligonucleotide probe containing sequenceshomologous to subtilisin from another strain of bacteria or fungus couldbe used as a probe to identify subtilisin-encoding clones, usinghybridization and washing conditions of lower stringency.

Yet another method for identifying subtilisin-producing clones wouldinvolve inserting fragments of genomic DNA into an expression vector,such as a plasmid, transforming protease-negative bacteria with theresulting genomic DNA library, and then plating the transformed bacteriaonto agar containing a substrate for subtilisin, such as skim-milk.Those bacteria containing subtilisin-bearing plasmid will producecolonies surrounded by a halo of clear agar, due to digestion of theskim-milk by excreted subtilisin.

Generation Of Site Directed Mutations In The Subtilisin Gene

Once the subtilisin gene has been cloned, and desirable sites formutagenesis identified, the mutations can be introduced using syntheticoligonucleotides. These oligonucleotides contain nucleotide sequencesflanking the desired mutation sites, mutant nucleotides are insertedduring oligonucleotide synthesis. In a preferred method, a singlestranded gap of DNA, bridging the subtilisin gene, is created in avector bearing the subtilisin gene. Then the synthetic nucleotide,bearing the desired mutation, is annealed to a homologous portion of thesingle-stranded DNA. The remaining gap is then filled in by DNApolymerase I (Klenow fragment) and the construct is ligated using T4ligase. A specific example of this method is described Morinaga et al.(1984); Biotechnology, 2 646-639!. According to Morinaga et al., afragment within the gene is removed using restriction endonuclease. Thevector/gene, now containing a gap, is then denatured and hybridized to avector/gene which, instead of containing a gap, has been cleaved withanother restriction endonuclease at a site outside the area involved inthe gap. A single-stranded region of the gene is then available forhybridization with mutated oligonucleotides, the remaining gap is filledin by the Klenow fragment of DNA polymerase I, the insertions areligated with T4 DNA ligase, and, after one cycle of replication, adouble-stranded plasmid bearing the desired mutation is produced. TheMorinaga method obviates the additional manipulation of constructing newrestriction sites, and, therefore, facilitates the generation ofmutations at multiple sites. U.S. Pat. No. 4,760,025, by Estell et al.,issued Jul. 26, 1988, is able to introduce oligonucleotides bearingmultiple mutations by performing minor alterations of the cassette,however, an even greater variety of mutations can be introduced at anyone time by the Morinaga method, because a multitude ofoligonucleotides, of various lengths, can be introduced.

Expression Of Subtilisin Variants

According to the invention, a mutated subtilisin gene produced bymethods described above, by the method described in Example 1, or anyalternative methods known in the art, can be expressed, in enzyme form,using an expression vector. An expression vector generally falls underthe definition of a cloning vector, since an expression vector usuallyincludes the components of a typical cloning vector, namely, an elementthat permits autonomous replication of the vector in a microorganismindependent of the genome of the microorganism, and one or morephenotypic markers for selection purposes. An expression vector includescontrol sequences encoding a promoter, operator, ribosome binding site,translation initiation signal, and, optionally, a repressor gene orvarious activator genes.

To permit the secretion of the expressed protein, nucleotides encoding a"signal sequence" may be inserted prior to the coding sequence of thegene. For expression under the direction of control sequences, a targetgene to be treated according to the invention is operably linked to thecontrol sequences in the proper reading frame. Promoter sequences thatcan be incorporated into plasmid vectors, and which can support thetranscription of the mutant subtilisin gene, include but are not limitedto the prokaryotic B-lactamase promoter Villa-Kamaroff, et al. (1978);Proc. Natl. Acad. Sci. U.S.A.; 75 3727-3731! and the tac promoterDeBoer, et al. (1983); Proc. NatI. Acad. Sci. U.S.A.; 80 21-25!. Furtherreferences can also be found in "Useful proteins from recombinantbacteria" in Scientific American (1980); 242 74-94.

According to one embodiment, B. subtilis is transformed by an expressionvector carrying the mutated DNA. If expression is to take place in asecreting microorganism such as B. subtilis a signal sequence may followthe translation initiation signal and precede the DNA sequence ofinterest. The signal sequence acts to transport the expression productto the cell wall where it is cleaved from the product upon secretion.The term "control sequences" as defined above is intended to include asignal sequence, when it is present.

The microorganisms able to produce a stabilized enzyme of this inventioncan be cultivated by conventional fermentation methods in a nutrientmedium containing assimilable carbon and nitrogen together with otheressential nutrients, the medium being composed in accordance with theprinciples of the known art.

Nucleotide Sequences And Microorganisms

This invention also relates to DNA nucleotide sequences encoding astabilized protease of the invention, and to an expression vectorcontaining a DNA nucleotide sequence encoding a stabilized protease.

The stabilized protease may be expressed and produced when DNAnucleotide sequence encoding this protease is inserted into a suitableexpression vector in a suitable host organism. The host organism is notnecessarily identical to the organism from which the parent geneoriginated. The construction of the mutated genes, vectors and mutantand transformed microorganisms may be carried out by any appropriaterecombinant DNA technique, known in the art.

The invention also relates to host organisms containing an expressionvector carrying a DNA nucleotide sequence encoding a stabilizedprotease.

Detergent Compositions

The present invention also comprises the use of the stabilized proteasesof the invention in cleaning and detergent compositions and suchcomposition comprising the stabilized proteases.

Such compositions comprise any one or more of the proteases of theinvention alone or in combination with any of the usual componentsincluded in such compositions which are well-known to the person skilledin the art.

Such components comprise builders, such as phosphate or zeolitebuilders, surfactants, such as anionic, cationic, non-ionic orzwitterionic type surfactants, polymers, such as acrylic or equivalentpolymers, bleach systems, such as perborate- or amino-containing bleachprecursors or activators, structurants, such as silicate structurants,alkali or acid to adjust pH, humectants, and/or neutral inorganic salts.

The detergent compositions of the invention can be formulated in anyconvenient form such as powders, liquids, etc.

The enzymes can be used in well-known standard amounts in detergentcompositions. The amounts may range very widely, e.g. about 0.0002-0.01,e.g. about 0.005-0.05, Anson units per gram of the detergentcomposition. Expressed in alternative units, the protease can beincluded in the compositions in amounts in the order of from about 0.1to 100 GU/mg (e.g. 1-50, especially 5-20 GU/mg) of the detergentformulation, or any amount in a wide range centering at about 0.01-4,e.g. 0.1-0.4 KNPU per g of detergent formulation.

The KNPU has been defined previously. A GU is a Glycine Unit, defined asthe proteolytic enzyme activity which, under standard conditions, duringa 15-minutes' incubation at 40 deg C., with N-acetyl casein assubstrate, produces an amount of NH2-group equivalent to 1 micromole ofglycine.

It may for example be suitable to use the present enzymes at the rate ofabout 0.25 mg of enzyme protein per liter of wash liquor, correspondingto an enzyme activity of the order of 0.08 KNPU per liter. Correspondingdetergent formulations can contain the enzymes in for example an amountof the order of 0.1-0.4 KNPU/g.

The detergent compositions may also contain further enzymes.

For example, lipase can usefully be added in the form of a granularcomposition (alternatively a solution or a slurry) of lipolytic enzymewith carrier material (e.g. as in EP Patent Publication No. 258,068(Novo Nordisk ANS) and the Lipolase™ and other enzyme compositions ofNovo Nordisk A/S).

The added amount of lipase can be chosen within wide limits, for example50 to 30,000 LU/g per gram of the surfactant system or of the detergentcomposition, e.g. often at least 100 LU/g, very usefully at least 500LU/g, sometimes preferably above 1000, above 2000 LU/g or above 4000LU/g or more, thus very often within the range of 50-4000 LU/g, andpossibly within the range of 200-1000 LU/g. In this specification,lipase units are defined as they are in EP Patent Publication No.258,068.

The lipolytic enzyme can be chosen among a wide range of lipases. Inparticular, the lipases described in for example the following patentspecifications: EP Patent Publications Nos. 214,761 (Novo Nordisk A/S),258,068, and especially lipases showing immunological crossreactivitywith antisera raised against lipase from Thermomyces lanuginosus ATCC22070, EP Patent Publications Nos. 205,208 and 206,390, and especiallylipases showing immunological cross-reactivity with antisera raisedagainst lipase from Chromobacter viscosum var lipolyticum NRRL B-3673,or against lipase from Alcaligenes PL-679, ATCC 31371 and FERM-P 3783,also the lipases described in specifications WO 87/00859 (Gist-Brocades)and EP Patent Publication No. 204,284 (Sapporo Breweries). Suitable, inparticular, are for example the following commercially available lipasepreparations: Lipolase Novo Nordisk ANS, Amano lipases CE, P, B, AP,M-AP, AML, and CES, and Meito lipases MY-30, OF, and PL, also EsteraseMM, Lipozym, SP225, SP285, Saiken lipase, Enzeco lipase, Toyo Jozolipase and Diosynth lipase (Trade Marks).

Amylase can for example be used when desired, in an amount in the rangeof about 1 to about 100 MU (maltose units) per gram of detergentcomposition (or 0.014-1.4, e.g. 0.07-0.7, KNU/g (Novo units)). Cellulasecan for example be used when desired, in an amount in the range of about0.3 to about 35 CEVU units per gram of the detergent composition.

Among the usual detergent ingredients which may be present in usualamounts in the detergent compositions of this invention are thefollowing: The compositions may be built or unbuilt, and may be of thezero-P type (i.e. not containing any phosphorus containing builders).Thus, the composition may contain in the aggregate for example from1-50%, e.g. at least about 5% and often up to about 35-40% by weight, ofone or more organic and/or inorganic builders. Typical examples ofbuilders include those already mentioned above, and more broadly includealkali metal ortho, pyro, and tripolyphosphates, alkali metalcarbonates, either alone or in admixture with calcite, alkali metalcitrates, alkali metal nitrilotriacetates, carboxymethyloxysuccinates,zeolites, polyacetalcarboxylates, and so on.

Furthermore, the detergent compositions may contain from 1-35% of ableaching agent or a bleach precursor or a system comprising bleachingagent and/or precursor with activator therefor. Further optionalingredients are lather boosters, foam depressors, anti-corrosion agents,soil-suspending agents, sequestering agents, anti-soil redepositionagents, perfumes, dyes, stabilising agents for the enzymes, and so on.

The compositions can be used for the washing of textile materials,especially, but without limitation cotton and polyesterbased textilesand mixtures thereof. For example washing processes carried out attemperatures of about 60°-65° C. or lower, e.g. about 30°-35° C. orlower, are particularly suitable. It can be very suitable to use thecompositions at a rate sufficient to provide about e.g. 0.4-0.8 g/l ofsurfactant in the wash liquor, although it is of course possible to uselower or higher concentrations, if desired. Without limitation it canfor example be stated that a use-rate from about 1 to 10 g/l, e.g. fromabout 3-6 g/l, of the detergent formulation is suitable for use in thecase when the formulations are substantially as in the Examples.

In some useful embodiments, the detergent compositions can be formulatedas follows:

Detergent I

A detergent powder according to an embodiment of the inventioncontaining zeolite builder is formulated to contain:

Total active detergent of about 16%, anionic detergent of about 9%,nonionic detergent of about 6%, zeolite-containing builder of about 20%,acrylic or equivalent polymer of about 3.5%, perborate bleach precursorof about 6-18%, amino-containing bleach activator of about 2%, silicateor other structurant of about 3.5%, alternatively down to about 2.5%,enzyme of about 8 (alternatively of about 15) glycine units/mg grade,with alkali to adjust to desired pH in use, and neutral inorganic salt,and enzymes (about 0.5% each enzyme).

The anionic detergent is a mixture of sodium dodecyl-benzene sulphonate,alternatively sodium linear alkyl-benzene-sulphonate, 6% and primaryalkyl sulphate of 3%. The nonionic detergent is an ethoxylate of anapprox. C13-C15 primary alcohol with 7 ethoxylate residues per mole. Thezeolite builder is type A zeolite. The polymer is polyacrylic acid. Theperborate bleach precursor is sodium tetraborate tetrahydrate ormonohydrate. The activator is tetraacetyl-ethylenediamine. Thestructurant is sodium silicate. The neutral inorganic salt is sodiumsulphate.

Detergent II

An aqueous detergent liquid according to an embodiment of the inventionis formulated to contain:

Dodecylbenzene-sulphonic acid of 16%, C12-C15 linear alcohol condensedwith 7 mol/mol ethylene oxide of 7%, monoethanolamine of 2%, citric acidof 6.5%, sodium xylenesulphonate of 6%, sodium hydroxide of about 4.1%,protease of 0.5%, minors and water to 100%. The pH is adjusted to avalue between 9 and 10.

In other useful embodiments, the detergent compositions can beformulated as e.g. in International Patent Publications Nos. WO91/00334, WO 91/00335, and International Patent Application No.PCT/DK91/00399.

The invention is further illustrated in the following examples, whichare not intended to be in any way limiting to the scope of the inventionas claimed.

EXAMPLE 1 Preparation Example

In the following Example, showing a presently preferred method forconstructing and expressing genes to code for wild-type and variantprotease enzymes in accordance with embodiments of the presentinvention, the following materials are referred to:

B. subtilis 309 and 147 are variants of Bacillus lentus, deposited withthe NCIB and accorded the accession numbers NCIB 10147 and NCIB 10309,and described in U.S. Pat. No. 3,723,250 incorporated by referenceherein.

E. coli MC 1000 (M. J. Casadaban and S. N. Cohen (1980); J. Mol. Biol.;138 179-207), was made r-,m+ by conventional methods and is alsodescribed in U.S. patent application Ser. No. 039,298.

A vector suited to a synthetic gene coding for subtilisin 309 and itsmutants was constructed. It is essentially a pUC19 plasmid C.Yanish-Perron and J. Messing (1985); Gene; 33 103-119!, in which themultiple cloning site has been replaced by a linker containing therestriction sites used to separate the five subfragments constitutingthe gene. The new linker was inserted into Eco RI--HindIll cut pUC19thereby destroying these sites. ##STR1##

A synthetic gene coding for the mature part of subtilisin 309 wasconstructed as shown by the following description and the diagrams givenin FIG. 1 (sheets 1/7 to 7/7) of the accompanying drawings. Thestructure of the synthetic gene is summarised in sheets 1/7 to 4/7,which also indicate fragments used in the construction. Each subfragmentwas made from 6 to 12 oligonucleotides. The oligonucleotides weresynthesised on an automatic DNA synthesizer using phosphoramiditechemistry on a controlled glass support S. L. Beaucage and M. H.Carruthers (1981); Tetrahedron Letters; 22 1859-1869!. Dots in the5'-end of the oligonucleotides in the Figures are meant to indicate thatthese oligonucleotides have been phosphorylated. Duplexes (indicated insheets 1/7 to 4/7) were formed from corresponding pairs ofoligonucleotides by heating for 5 min at 90 deg C. followed by coolingto room temperature over a period of 75 min. The duplexes were mixed andtreated with T4 DNA ligase.

The five subfragments were isolated on a 2% agarose gel and insertedinto pSX191. The sequence was verified by dideoxynucleotide sequencing.Fragments A-E were isolated and ligated together with KpnI-BamHI cutpSX191. The ligation mixtures were used to transform competent E coliMC1000 r-,m+ selecting for ampicillin resistance. The 850 bp KpnI-BamHIfragment that constitutes the part of the subtilisin 309 gene coding forthe mature part of the enzyme was then used to replace the wild typegene on pSX212 giving rise to pSX222, which was then transformed intocompetent B subtilis SHa273. After fermentation of the transformedstrain and purification of the enzyme it was shown that the product wasindistinguishable from the wild type product.

Protease variants derived from the synthetic gene are made by usingoligonucleotides with altered sequence at the place(s) where mutation iswanted (e.g. with sequences as given below) and mixing them with therest of the oligonucleotides appropriate to the synthetic gene. Assemblyof the variant gene is carried out with the variant materials in amanner otherwise analogous to that described above. Further informationon synthetic genes generally is available in Agarval et al (1970);Nature; 227 27-34.

A KpnI site was introduced into the beginning of the subtilisin 309synthetic gene encoding the mature part of the enzyme. The method usedis called oligonucleotide directed double-strand break repairmutagenesis and is described by Wlodek Mandecki (1986); Proc. Nat. Acad.Sci. USA; 83 7177-7181. pSX1 72 is opened with NcoI at the beginning ofthe mature part of the subtilisin 309 gene and is mixed with theoligonucleotide NOR 789 (sequence shown in FIG. 1 (7/7)), heated to 100deg C., cooled to 0 deg C., and transformed into E coli. Afterretransformation, the recombinants can be screened by colonyhybridisation using 32-P-labelled NOR 789. The recombinants that turnedout to be positive during the screening had the KpnI site introducedright in front of NcoI by changing two bases without changing the aminoacid sequence. pSX172 is described in EP Patent Publication No. 405,901.The KpnI site so created is inserted into pSX120 on a 400-bp PvuI-NheIfragment, giving rise to pSX212. pSX120 is also described in EP PatentPublication No. 405,901.

The synthetic gene is inserted between KpnI and BamHI on pSX212, givingrise to pSX222.

Examples of mutations and corresponding sequences of oligonucleotidesare as follows: ##STR2##

These oligonucleotides were combined with the rest of theoligonucleotides from the synthetic gene that was not changed.

EXAMPLE 2 Purification Example

This procedure relates to purification of a 10 liter scale fermentationof the Subtilisin 147 enzyme, the Subtilisin 309 enzyme or mutantsthereof.

Approximately 8 liters of fermentation broth were centrifuged at 5000rpm for 35 minutes in 1 liter beakers. The supernatants were adjusted topH 6.5 using 10% acetic acid and filtered on Seitz Supra S100 filterplates.

The filtrates were concentrated to approximately 400 ml using an AmiconCH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UFconcentrate was centrifuged and filtered prior to absorption at roomtemperature on a Bacitracin affinity column at pH 7. The protease waseluted from the Bacitracin column at room temperature using 25%2-propanol and 1M sodium chloride in a buffer solution with 0.01dimethylglutaric acid, 0.1M boric acid and 0.002M calcium chlorideadjusted to pH 7.

The fractions with protease activity from the Bacitracin purificationstep were combined and applied to a 750 ml Sephadex G25 column (5 cmdia.) equilibrated with a buffer containing 0.01 dimethylglutaric acid,0.2M boric acid and 0.002 m calcium chloride adjusted to pH 6.5.

Fractions with proteolytic activity from the Sephadex G25 column werecombined and applied to a 150 ml CM Sepharose CL 6B cation exchangecolumn (5 cm dia.) equilibrated with a buffer containing 0.01Mdimethylglutaric acid, 0.2M boric acid, and 0.002M calcium chlorideadjusted to pH 6.5.

The protease was eluted using a linear gradient of 0-0.1M sodiumchloride in 2 liters of the same buffer (0-0.2M sodium chloride in caseof sub 147).

In a final purification step protease containing fractions from the CMSepharose column were combined and concentrated in an Amiconultrafiltration cell equipped with a GR81PP membrane (from the DanishSugar Factories Inc.).

EXAMPLE 3 Differential Scanning Calorimetry

The purified protease variants were subjected to thermal analysis byDifferential Scanning Calorimetry (DSC).

The instrument was a Setaram micro DSC apparatus connected to HP86computer for data collection and analysis. Setaram software was used.

The enzyme was diluted to a concentration of preferably 2 mg/ml in aliquid built detergent (pH 8.5) of the following composition:

    ______________________________________    AE.sup.1 (C.sub.12-14); EO 6                            15%    LAS.sup.2 (C.sub.12)    10%    Coconut fatty acid      9%    Oleic acid (C.sub.18)   1%    Triethanolamin (pKA 7.9)                            9%    Glycerol                10.5%    Ethanol                 1.5%    Sodium citrate          8%    CaCl; 2H.sub.2 O        0.1%    NaOH                    1%    Water                   34.9%    ______________________________________     .sup.1 Alcohol ethoxylate     .sup.2 Linear alkylbenzene sulphonate

The heating rate was 0.5° C./min from 25° C. to 90° C.

The stabilization of Subtilisin 309 variants relative to wild typeenzyme measured by this method is presented in Table 3, above.

EXAMPLE 4 Storage Stability

The storage stability of the enzymes of the invention was determined andcompared to the storage stability of subtilisin 309 (wild-type). Thisstability test is performed as a Mini Storage Test. In each tube 100 μlof sample were used.

The enzyme dosages were 0.25 mg enzyme/g detergent. A liquid detergentcomposition was used in this test, vide Detergent II, supra.

The tubes were incubated at 35° C. for 3, 7, 14, and 21 days,respectively, and the residual activity determined.

The residual activity determination method was based on the digestion ofa dimethyl casein (DMC) solution by the proteolytic enzymes. The primaryamino groups formed in this process react with trinitrobenzene sulphonicacid (TNBS) forming a coloured complex. The reaction is followed in situin order that the change in absorbance per time unit can be calculated.

Since the detergent might contain compounds with primary amino groups,it is necessary to examine this and make correction of the detergenteffect. Correction is made by measuring the blind value of the puredetergent and subtracting this value from the value measured asdescribed above.

The activity is determined relative to a sample which immediately afterpreparation is frozen and kept at a temperature of -10° C. untilanalysis. The activity of this sample is set to 100%. The activity ofthe corresponding samples from the storage test is determined relativeto the 100% sample.

The reaction conditions were:

    ______________________________________    Temperature:   40° C.    pH:            8.3    Wavelength:    420 nm    Reaction time: 9 min.    Measuring time:                   3 min.    Apparatus:     <COBAS> FARA II centrifugal ana-                   lyzer from Roche.    ______________________________________

All activities were determined in duplicate.

A folder, AF 285/1 (or later editions), describing this analyticalmethod is available upon request to Novo Nordisk A/S, Denmark, whichfolder is hereby included by reference.

The result of the storage stability test is shown in FIG. 2. In general,thermostability as determined by DSC and storage stability correlatewell.

EXAMPLE 5 Wash Performance

The wash performance tests were accomplished on grass juice soiledcotton in a model wash at 20° C., isothermically for 10 minutes.

As detergent 5 g/l of a powder detergent was used, vide Detergent I,supra. pH was adjusted by addition of NaOH/HCl to 10.2. The water usedwas approximately 9° dH (German Hardness) for the tests presented inTable 5. For the tests presented in Table 6, 6° dH water was used. Thetextile/wash liquor ratio was 6 g textile per liter of wash liquor.

Tests were performed at enzyme concentrations of: 0, 0.025, 0.05, 0.1,0.5, 1.0, and 2.0 mg enzyme protein/I. Two independent sets of testswere performed for each of the enzymes. The results shown in Tables 5-6are means of these tests.

Subsequent to washing, the fabric was rinsed in running tap water andair-dried. The protease performance was determined by the change (ΔR) ofthe remission (% R) at 460 nm measured on a Datacolor Elrephometer 2000,ΔR being the remission after wash with protease added minus theremission after wash with no protease added.

Results from the wash performance tests are presented in Tables 5-6. Itis found that all the variants, except Subtilisin 309/S256P, perform atleast equal to subtilisin 309 (wild-type).

                  TABLE 5    ______________________________________    Differential Remission, delta R           Enzyme dosage mg enzyme/l detergent    Variant  0.025  0.05     0.1  0.5    1.0  2.0    ______________________________________    S57P     2.4    4.4      7.4  16.8   20.0 22.1    A172P    2.4    4.4      7.5  16.9   20.1 22.3    S188P    2.2    4.8      7.5  16.9   20.9 21.7    A194P    2.5    5.3      7.6  17.5   20.5 20.8    Subt. 309             3.2    4.6      6.9  16.1   20.4 20.7    S259P    1.7    2.5      3.9  6.7    7.2  7.0    Subt. 309             0.8    2.2      3.6  5.8    6.5  6.6    ______________________________________

                  TABLE 6    ______________________________________    Differential Remission, delta R           Enzyme dosage mg enzyme/l detergent    Variant  0.025  0.05     0.1  0.5    1.0  2.0    ______________________________________    T38P     1.9    3.7      7.0  14.7   16.9 16.7    S99P     1.8    3.1      5.4  15.4   16.4 16.9    S256P    0.7    1.5      3.0  9.7    15.2 16.8    Subt. 309             1.9    3.9      6.9  14.5   16.2 17.1    ______________________________________

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 10    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 47 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    AATTGGTACCCTGCAGGAATTCAAGCTTATCGATGGCATGCGGATCC47    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 47 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    AGCTGGATCCGCATGCCATCGATAAGCTTGAATTCCTGCAGGGTACC47    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    AACAACCGCGCTAGCTTTTCACAGTATGGCCCAGGC36    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    GTCAAGGCCTGGGCCATACTGTGAAAAGCTAGCGCG36    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 42 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    CGCTATCCGAACGCAATGGCAGTCGGAGCTACTGATCAAAAC42    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 42 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GTTGTTGTTTTGATCAGTAGCTCCGACTGCCATTGCGTTCGG42    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    AGCTTTGTACCAGGGGAACCGCCGACTCAAGATGGG36    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 38 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    CCCATTCCCATCTTGAGTCGGCGGTTCCCCTGGTACAA38    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 275 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    AlaGlnSerValProTyrGlyValSerGlnIleLysAlaProAlaLeu    151015    HisSerGlnGlyTyrThrGlySerAsnValLysValAlaValIleAsp    202530    SerGlyIleAspSerSerHisProAspLeuLysValAlaGlyGlyAla    354045    SerMetValProSerGluThrAsnProPheGlnAspAsnAsnSerHis    505560    GlyThrHisValAlaGlyThrValAlaAlaLeuAsnAsnSerIleGly    65707580    ValLeuGlyValAlaProSerAlaSerLeuTyrAlaValLysValLeu    859095    GlyAlaAspGlySerGlyGlnTyrSerTrpIleIleAsnGlyIleGlu    100105110    TrpAlaIleAlaAsnAsnMetAspValIleAsnMetSerLeuGlyGly    115120125    ProSerGlySerAlaAlaLeuLysAlaAlaValAspLysAlaValAla    130135140    SerGlyValValValValAlaAlaAlaGlyAsnGluGlyThrSerGly    145150155160    SerSerSerThrValGlyTyrProGlyLysTyrProSerValIleAla    165170175    ValGlyAlaValAspSerSerAsnGlnArgAlaSerPheSerSerVal    180185190    GlyProGluLeuAspValMetAlaProGlyValSerIleGlnSerThr    195200205    LeuProGlyAsnLysTyrGlyAlaTyrAsnGlyThrSerMetAlaSer    210215220    ProHisValAlaGlyAlaAlaAlaLeuIleLeuSerLysHisProAsn    225230235240    TrpThrAsnThrGlnValArgSerSerLeuGluAsnThrThrThrLys    245250255    LeuGlyAspSerPheTyrTyrGlyLysGlyLeuIleAsnValGlnAla    260265270    AlaAlaGln    275    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 269 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    AlaGlnSerValProTrpGlyIleSerArgValGlnAlaProAlaAla    151015    HisAsnArgGlyLeuThrGlySerGlyValLysValAlaValLeuAsp    202530    ThrGlyIleSerThrHisProAspLeuAsnIleArgGlyGlyAlaSer    354045    PheValProGlyGluProSerThrGlnAspGlyAsnGlyHisGlyThr    505560    HisValAlaGlyThrIleAlaAlaLeuAsnAsnSerIleGlyValLeu    65707580    GlyValAlaProSerAlaGluLeuTyrAlaValLysValLeuGlyAla    859095    SerGlySerGlySerValSerSerIleAlaGlnGlyLeuGluTrpAla    100105110    GlyAsnAsnGlyMetHisValAlaAsnLeuSerLeuGlySerProSer    115120125    ProSerAlaThrLeuGluGlnAlaValAsnSerAlaThrSerArgGly    130135140    ValLeuValValAlaAlaSerGlyAsnSerGlyAlaGlySerIleSer    145150155160    TyrProAlaArgTyrAlaAsnAlaMetAlaValGlyAlaThrAspGln    165170175    AsnAsnAsnArgAlaSerPheSerGlnTyrGlyAlaGlyLeuAspIle    180185190    ValAlaProGlyValAsnValGlnSerThrTyrProGlySerThrTyr    195200205    AlaSerLeuAsnGlyThrSerMetAlaThrProHisValAlaGlyAla    210215220    AlaAlaLeuValLysGlnLysAsnProSerTrpSerAsnValGlnIle    225230235240    ArgAsnHisLeuLysAsnThrAlaThrSerLeuGlySerThrAsnLeu    245250255    TyrGlySerGlyLeuValAsnAlaGluAlaAlaThrArg    260265    __________________________________________________________________________

We claim:
 1. A substantially pure stabilized subtilisin, in which theamino acid proline is substituted for a naturally occurring amino acidand which position(s) is/are not located in regions, in which theprotease is characterized by possessing α-helical or β-sheet structure.2. The stabilized subtilisin of claim 1 wherein the dihedral anglesconstitute values within the intervals -90°<φ<-40° and 120°<ψ<180°. 3.The stabilized subtilisin of claim 1 wherein the dihedral anglesconstitute values within the intervals -90°<φ<40° and -50°<ψ<10°.
 4. Thesubtilisin of claim 1, further comprising one or more of the followingBPN substitutions: 27R, 36D, 97N, 98R, 188P, 194P, 120D, 128G, 195E,235L, 235R, 237R, 251E, and 263F.
 5. The subtilisin of claim 1, selectedfrom the group consisting of: a stabilized subtilisin 309, a stabilizedsubtilisin 147, a stabilized subtilisin BPN', and a stabilizedsubtilisin Carlsberg.
 6. The stabilized subtilisin 309 of claim 5 havingone or more proline substitutions at positions numbered according totheir alignment with the analogous amino acid position in the amino acidsequence of the mature subtilisin BPN selected from the group consistingof other than proline at one or more positions selected from the groupconsisting of 38, 57, 98, 172, 242 and 259, said positions numberedaccording to their alignment with the analogous amino acid position inthe amino acid sequence of the mature subtilisin BPN, wherein thenaturally occurring amino acid to be substituted at said one or morepositions has values for the dihedral angles φ (phi) and ψ (psi) withinthe interval -90°<φ<-40° and within the interval -180°<ψ<180° T38P,S57P, A98P, A172P, S188P, A194P, S242P, and S259P.
 7. Subtilisin 309 ofclaim 6, further comprising one or more of the following BPNsubstitutions: K27R, *36D, N76D, G97N, A98R, V104Y, H120D, S128G, G195E,Q206C, N218S, K235L, K235R, K237R, K251E, and Y263F.
 8. A substantiallypure, stabilized, subtilisin 309 having the following amino acidsubstitutions at positions numbered according to their alignment withthe analogous amino acid position in the amino acid sequence of themature subtilisin BPN K27R+*36D+G97N+A98R+A194P+K235R+K237R+K251E+Y263F.9. A substantially pure, stabilized, subtilisin 309 having the followingamino acid substitutions at positions numbered according to theiralignment with the analogous amino acid position in the amino acidsequence of the mature subtilisin BPNK27R+*36D+G97N+A194P+K235R+K237R+K25 E+Y263F.
 10. A substantially pure,stabilized, subtilisin 309 having the following amino acid substitutionsat positions numbered according to their alignment with the analogousamino acid position in the amino acid sequence of the mature subtilisinBPN K27R+*36D+G97N+A194P+K235R+K237R+Y263F.
 11. A substantially pure,stabilized, subtilisin 309 having the following amino acid substitutionsat positions numbered according to their alignment with the analogousamino acid position in the amino acid sequence of the mature subtilisinBPN *36D+G97N+V104Y+H120D+A194P+G195E.
 12. A substantially pure,stabilized, subtilisin 309 having the following amino acid substitutionsat positions numbered according to their alignment with the analogousamino acid position in the amino acid sequence of the mature subtilisinBPN *36D+G97N+v104Y+H120D+A194P+G195E+K235L.
 13. A substantially pure,stabilized, subtilisin 309 having the following amino acid substitutionsat positions numbered according to their alignment with the analogousamino acid position in the amino acid sequence of the mature subtilisinBPN *36D+G97N+H120D+A194P+G195E.
 14. A substantially pure, stabilized,subtilisin 309 having the following amino acid substitutions atpositions numbered according to their alignment with the analogous aminoacid position in the amino acid sequence of the mature subtilisin BPN*36D+G97N+H120D+Al94P+G195E+K235L.
 15. A substantially pure, stabilized,subtilisin 309 having the following amino acid substitutions atpositions numbered according to their alignment with the analogous aminoacid position in the amino acid sequence of the mature subtilisin BPN*36D+V104Y+H120D+A194P+G195E.
 16. A substantially pure, stabilized,subtilisin 309 having the following amino acid substitutions atpositions numbered according to their alignment with the analogous aminoacid position in the amino acid sequence of the mature subtilisin BPN*36D+V104Y+H120D+A194P+G195E+K235L.
 17. A nucleotide sequence encoding astabilized protease of claim
 1. 18. An expression vector comprising anucleotide sequence encoding a stabilized protease according to claim 1.19. A host organism containing an expression vector carrying anucleotide sequence encoding a stabilized protease of claim 1.